This invention relates to a process for producing heparinase utilizing a defined medium followed by a purification step.
Heparinase is an enzyme presently used in assays for heparin. Presently, heparinase is produced from Flavobacterium heparinum utilizing protein as the carbon, and nitrogen source and a phosphate source. In addition, presently available processes for producing heparinase provide a volumetric productivity of heparinase of about 1.8 mg heparin degrade/liter culture--hour. Furthermore, these prior processes require six stages of production including: (1) 72 hour fermentation; (2) Harvest by centrifugation; (3) 24 hour fermentation; (4) Harvest by centrifugation; (5) 17 hour incubation and (6) Harvest and sonication. In present processes, the crude enzyme produced by the bacterium is isolated by a step which includes passage of the crude enzyme through a column of hydroxylapatite (Ca.sub.x) (PO.sub.4)y). It has been found that the heparinase binds more tightly to the hydroxylapatite than 90% of the protein present in Flavobacterium heparinum. Thus, a hydroxylapatite column can provide for 10-100 fold enzyme enrichment when the protein is eluted from th column at high eluent salt concentrations. However, hydroxylapatite is fragile and has poor flow characteristics in large columns and thus is not amenable to purification of much more than 1-10 grams of crude enzyme. Furthermore, each such purification requires nearly a week's time.
Accordingly, it would be desirable to provide a process for purifying heparinase which is not limited by the materials and methods utilized in purifying heparinase and which require far less time than required by present available processes. In addition, it would be desirable to provide such a process which drastically increases the volumetric productivity of heparinase.